Here we seek to understand the role of RNA interference (RNAi) machinery in mammalian antiviral defense. RNAi is a nucleic acid sequence-specific means of gene silencing guided by short, double-stranded RNA. Plants, insects and C. elegans use RNAi to combat viral infections, however mammals evolved with alternative antiviral mechanisms, including pattern recognition receptors (PRRs) and components of the inflammasome, leading to the production of type I IFN and other cytokines to achieve an antiviral state. The key RNAi effector proteins, Dicer and Argonautes (AGO)1-4 are functional in mammalian cells, being essential for development and regulation of the microRNA and RNAi systems. However, any role for RNAi machinery in mammalian host- virus interactions remains to be fully characterized. In addition, whether redundancy between the four mammalian AGOs exits, for executing RNAi or other small RNA-mediated repression in any physiological context remains unclear. This work is significant because this ancient mechanism of innate immunity may be important for defense against many human viruses. To study the role of RNAi machinery in mammalian immunology, we will use mice lacking individual AGO proteins, which will serve as primary tools for the proposed studies. In preliminary results reported here, we demonstrate profound but differential contributions of AGOs 2 and 4 to mammalian antiviral responses. Antiviral responses are markedly dimished in the absence of AGO2 catalytic activity or AGO4 protein. Moreover, AGO1 and 3 appear to play minimal roles in antiviral defense. However, a lack of AGO2 protein resulted in reduced virus levels, suggesting AGO2 negatively regulates other antiviral pathways. This application aims to comprehensively dissect the functions of individual Argonautes in the antiviral response. Specifically, the studies proposed in aim 1 will characterize the role of AGO2, the only argonaute with slicing capacity, in silencing virus. Aim 2 proposes to decipher how AGO4 contributes to the mammalian antiviral responses. Both aims will determine whether AGOs act autonomously or how they aid or compete with existing PRR and type I IFN mammalian antiviral defense mechanisms. Aim 3 will assess what host and virus-derived RNA populations bind to AGOs 2 and 4 in influenza A infected macrophages. These virus-derived RNAs will then be tested for their virus silencing capacity. This work will elucidate the undefined roles of individual AGO proteins in antiviral defense and may offer new therapeutic targets for the treatment of viral infections.